Molecular and biological activities of metal oxide-modified bioactive glass

Bioactive glass (BG) was prepared by sol–gel method following the composition 60-(\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x$$\end{document}x) SiO2.34CaO.6P2O5, where x = 10 (FeO, CuO, ZnO or GeO). Samples were then studied with FTIR. Biological activities of the studied samples were processed with antibacterial test. Model molecules for different glass compositions were built and calculated with density functional theory at B3LYP/6-31 g(d) level. Some important parameters such as total dipole moment (TDM), HOMO/LUMO band gap energy (ΔE), and molecular electrostatic potential beside infrared spectra were calculated. Modeling data indicated that P4O10 vibrational characteristics are enhanced by the addition of SiO2.CaO due to electron rush resonating along whole crystal. FTIR results confirmed that the addition of ZnO to P4O10.SiO2.CaO significantly impacted the vibrational characteristics, unlike the other alternatives CuO, FeO and GeO that caused a smaller change in spectral indexing. The obtained values of TDM and ΔE indicated that P4O10.SiO2.CaO doped with ZnO is the most reactive composition. All the prepared BG composites showed antibacterial activity against three different pathogenic bacterial strains, with ZnO-doped BG demonstrating the highest antibacterial activity, confirming the molecular modeling calculations.

Recently, phosphate-based glasses have gained a lot of interest as local delivery systems for antimicrobial metal ion delivery due to their ability to dissolve at a constant rate, possessing a non-toxic nature, and having the ability to reach directly to the site of interest. This has led to their use for the development of new antimicrobial agents. Microbes in light of the rapid and continuous development of strains that are resistant to traditional drugs and antibiotics. When cobalt oxide, copper oxide and zinc oxide were added to phosphate glass (5 mg/ ml), it showed a strong antimicrobial activity against gram-positive and gram-negative bacteria 1 . Phosphate bioactive glasses doped with metal oxides (MO) have been widely investigated for their antimicrobial efficacy against a range of clinically significant microorganisms 2 . Bioactive glasses (BG) can be used to improve general health because they reduce the risk of bone and joint infections as antimicrobial biomaterials that are used in medical implants 3 . Recently, there has been much interest in focusing on MO nanoparticles because of their numerous applications and as bactericides 4,5 . Because of their abundance, good thermal stability, in addition to low biological toxicity and biodegradability, they are used in a wide variety of applications 6 . When iron oxide (Fe 2 O 3 ) is added to bioglass, it increases the density of the crosslinking, and makes it more durable by greatly reducing the rate of decomposition. It can be used to produce fibers of different diameters 2,7 . For example, it has been found that phosphate-containing glass fibers (PGF) supplemented with Fe 2 O 3 have applications such as being used as cell-delivery compounds for muscle stem cells in order to replace damaged or diseased muscles, and their biocompatibility can be assessed by using other types of cells 8 . When using BG containing Li 2 O-Fe 2 O 3 and studying the biological activity in the laboratory, the effect of these materials was noticed, as they represented new magnetic biomaterials used as a treatment for cancer with high temperature 9 . Compared with the iron-free glasses, Fe 2 O 3 -doped mesoporous BGs had the ability to increase the standard rate constant of Electro-Fenton's reaction up to 38.44, thus having the ability to produce reactive oxygen species, and can, thus, be very valuable in cancer therapy strategies 10 . It is well known that zinc is essential for all living organisms due to its role in www.nature.com/scientificreports/ many cellular processes. In addition, there are more than 200 enzymes that need zinc as a cofactor in order to carry out metabolic processes 11 . It was found that there is an antimicrobial efficacy of glass based on phosphate saturated with zinc in the treatment of urinary tract infections 12 . Phosphate glasses doped with zinc oxide at 5 mol% showed promising results for reducing antimicrobial resistance and host cell toxicity 1 . Cu-/Zn-doped borate mesoporous BGs prepared using cost-effective method showed great potential for wound healing/skin tissue engineering applications, associated with excellent antibacterial activity against Pseudomonas aeruginosa 13 . Metal ions can be present in the composition of BG (SiO 2 .P 2 O 5 .CaO) prepared by sol-gel method 14 . It was found that copper ions can be easily released from solid particles compared to ions of heavy elements. It can be inferred from this that the release of vital metal ions from the molecules confirms their antimicrobial effect 14 . It was found from the description of copper added to phosphate glass that it has antibacterial properties 8 . This was assessed by studying the effect of CuO and observing the speed of fiber withdrawal on the properties of the glass using fast differential scanning calorimetry (DSC), X-ray diffraction (XRD) and differential thermal analysis (DTA) 7 . The effect of increasing copper content in phosphate-based glass based on Na 2 O.CaO.P 2 O 5 -doped system on the viability of biofilm in vitro was studied 7,8 . Hollow BG nanoparticles enriched with copper and danofloxacin significantly decreased bacterial growth in sessile, planktonic and biofilm states 15 . Characterization of newly developed phosphate-based glass was done with investigating the effect of germanium (Ge) on the structure of SiO 2 .ZnO.CaO.SrO.P 2 O 5 glass and studying the subsequent effect on the formation of glass polyalkene cements and solubility, as well as bioactivity 16 . Phosphate-based glass with Ge additive (GeO 2 ) was developed to enhance nuclear radiation-shielding behaviors and mechanical properties 17 . Owing to the antimicrobial activity of Ge, GeO 2 was used to enhance the antibacterial properties of silicocarnotite bioceramic, where results showed effective antibacterial activity against Escherichia coli and Staphylococcus aureus 18 . Molecular modeling with different levels of theory is the most accurate method for predicting structural changes for given model molecules in response to their surrounding chemical environments 19 . It enables researchers to calculate infrared (IR) and Raman spectra with considerable precision 20,21 for a wide range of molecules. It could also predict some important physical parameters such as total dipole moment (TDM) and HOMO/LUMO band gap energy (ΔE) 22,23 . One can also utilize molecular modeling to investigate surface activity in terms of molecular electrostatic potential (MESP) 24 . Such class of computational work could be utilized for small clusters of atoms which can be successfully used also for glasses simulations 25 .
The aim of the present study is to prepare BG by sol-gel method following the composition 60-(x ) SiO 2 -34CaO-6P 2 O 5 were x = 10 (FeO, CuO, ZnO or GeO). Samples were studied with Fourier transform infrared spectroscopy (FTIR) then their biological activities were processed with antibacterial test. Model molecules for the different glass compositions were conducted with density functional theory (DFT) at B3LYP/6-31 g(d) level. Some important parameters such as TDM, molecular electrostatic potential, as well as infrared spectra were calculated.

Materials and methods
Calculations details. All the studied models were subjected to quantum mechanical calculations using GAUSSIAN 09 26 softcode at Molecular Spectroscopy and Modeling Unit, Spectroscopy Department, National Research Centre, Cairo Egypt. DFT at B3LYP/6-31 g(d) [27][28][29] level was used to calculate TDM, ΔE, MESP and IR frequencies. Partial density of states (PDOS) plots were generated using GaussSum 30  Methods. Synthesis of glass powder. Samples were prepared using the sol-gel method reported by Tohamy et al. 31 . The preparation of the gels involved using a quick alkali-mediated sol-gel method. This system consisted of four samples in addition to the control sample. BG was doped with [FeO, CuO, ZnO or GeO] (10 wt%). In the first step, TEOS was dissolved in ethanol/nitric acid solution and deionized H 2 O with continuous stirring for 45 min. Then, calcium nitrate tetrahydrate was added to the solution and stirred for 45 min. TEP was finally added to the solution and stirred for 45 min. After the final addition, the mixture of all reagents was left for www.nature.com/scientificreports/ 60 min to complete hydrolysis. Ammonia solution of 2 M concentration (a gelation catalyst) was dropped into the mixture. The mixture was then manually agitated with glass rod (as a mechanical stirrer) to prevent the formation of a bulk gel. Finally, each prepared gel was left to dry at 100-120 °C for 2 days and sintered at 600 °C for 2 h in thermal oven.
Antibacterial activity of prepared glass composites nanoparticles. The antibacterial activity of BG composites was determined using the paper disc diffusion assay reported earlier 32 . In brief, Tryptic Soy Agar (TSA) plates were prepared and inoculated with a 1 mL cell suspension of each bacterial pathogen separately, including gram-negative bacteria Pseudomonas aeruginosa (ATCC 10145) and Aeromonas hydrophila strain, and grampositive bacteria Staphylococcus aureus (ATCC 25923), obtained from the Microbial Inoculants Center-MIC, Ain Shams University, Egypt. Each pathogenic bacteria strain's pure culture was inoculated on TSA plates and incubated for 24 h at 37 °C. 4-5 loops from each strand were transferred into culture tubes containing 5 mL of sterile Tryptic Soy Broth (TSB) and incubated for 12 h at 37 °C. Mueller Hinton agar plates were inoculated with a 10 6 cell/mL microorganism suspension using cotton swabs. Sterile filter paper discs (Whatman ® Glass Microfibre filters, 6 mm in diameter) were impregnated with 20 µg/mL of various MO nanoparticle solutions.

Results and discussions
Molecular modeling. Building    www.nature.com/scientificreports/ Theoretical IR band assignments. The DFT:B3LYP/6-31 g(d) level was used to optimize then calculating vibrational spectra of the studied 6 model molecules. Figure 3 presents the calculated IR spectra for a-P  Correlating the above data, one can conclude that the vibrational characteristics of P 4 O 10 are enhanced by SiO 2 .CaO additive due to electron rush resonating along whole crystal. Additive ZnO to P 4 O 10 . SiO 2 .CaO highly impacted the vibrational characteristics in contradiction to the other alternatives CuO, FeO and GeO that caused minimal change in spectral indexing.
In order to experimentally verify the above findings, BG was prepared then characterized using FTIR. The experimental approach is needed to verify the molecular modeling data.
Molecular electrostatic potential mapping. A molecular electrostatic potential (MESP) map is useful threedimensional plot that demonstrates the distribution of electron density (charges) around the molecule and can, thus, be used to predict the charge-related properties of molecules, and to identify the site of electrophilic and nucleophilic attacks 33 . MESP map of a molecule's surface can be easily interpreted in terms of different colors, which are arranged from highest to lowest electron density (electronegativity) in the following order: red > orange > yellow > green > blue. Therefore, the lowest electrostatic potential is found in red regions, whereas the highest electrostatic potential is found in blue. Based on this and as seen in Fig. 4a, the oxygen atoms in the P 4 O 10 unit displayed higher electron density than phosphorus atoms. In Fig. 4b, significant change took place in the MESP map with the presence of SiO 2 and CaO with, again, oxygen atoms representing the regions of higher electronegativity.
Similar behavior can be clearly seen in Fig. 5 where CuO, FeO, ZnO and GeO metal oxide dopants resulted in significant change in the electron density distribution and electronegativity on the molecule's surface, thus introducing sites ready for nucleophilic attack and others ready for electrophilic attack.
The bacterial membranes are negatively charged due to the highly electronegative groups on membrane's phospholipids and lipopolysaccharides 34 . Therefore, it has been reported that the membrane may be the site of the antimicrobial activity of cationic metals attracted to it via electrostatic attraction [34][35][36] . It can, thus, be concluded that differences in electronegativity play a major role and has a direct effect on antimicrobial activity 37 .
Total dipole moment and HOMO/LUMO band gap energy. The dipole moments are highly sensitive even to small errors and are, therefore, an efficient check for the quality of computations, and in describing the overall electron density properties 38 . TDM is used to detect the nature of reactivity and impurity atoms effect of system, www.nature.com/scientificreports/ and it is well established in several studies that TDM is closely related to reactivity in such a way that the higher the TDM, the higher the reactivity [38][39][40] . Band gap energy is the difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) in the system. ΔE has been used as a simple indicator of the molecular chemical stability and could indicate the affinity pattern of the molecule 41 .
The calculated values of TDM and ΔE are listed in Table 2. The values demonstrated significant increase in the value of TDM in BG, measuring 27.1994 Debye, compared to P 4 O 10 for which the TDM was zero. ΔE was well correlated with TDM, showing significant decrease in its value to 1.7129 eV for BG, compared to 8.0268 eV for P 4 O 10 . This result indicates significant increase in the reactivity of the BG.
The results of TDM and ΔE energy after doping of BG with different MO indicated that all BG.MO structures experienced significant decrease in their TDM compared to BG, with ΔE also showing decrease in its value for all BG.MO structures. However, comparing the values of TDM and ΔE of the four BG.MO structures with one another, it is clear that BG.ZnO is the most reactive structure owing to its highest TDM and lowest ΔE. In addition, it has been reported that ZnO nanoparticles, interestingly, contain a positive charge in water suspensions 42 .  www.nature.com/scientificreports/ Correlating TDM and ΔE results with those of MESP mapping, and since the total bacterial charge is negative because of the negatively charged bacterial membranes, it can be concluded that BG.ZnO has a greater affinity to create electrostatic forces as a powerful bond with the bacterial surface, thus providing a stronger antibacterial effect 43 , which is confirmed by the assessment of antibacterial activity of the different glass composites, as later presented in Section "Assessment of antibacterial activity".
Partial density of states. In order to thoroughly determine the effect of the MOs on the electronic structure/ band gap of BG and the contribution of each metal, PDOS plots are depicted in Fig. 6. The wide ΔE of P 4 O 10 molecule, mentioned in Table 2, which reflects its low reactivity compared to the other structures, is clearly demonstrated in Fig. 6a. The atomic orbitals contribution of P and O in the PDOS plot of P 4 O 10 reflects that the atomic orbitals of both P and O contributed to the valence states, with the 2p atomic orbitals of O demonstrating higher contribution for the HOMO than the 3p atomic orbitals of P. Whereas, the LUMO of the conduction band indicates almost equal contribution by P and O atomic orbitals. In Fig. 6b depicting the PDOS of P 4 O 10 .SiO 2 .
CaO, ΔE showed significant decrease compared to that shown in P 4 O 10 plot. Also HOMO showed almost equal contributions of the atomic orbitals of O, Si and Ca, with very minimal contribution of the atomic orbitals of P, while LUMO is clearly dominated by the contribution of the atomic orbitals of Ca. The PDOS plot of BG CuO shown in Fig. 6c, the atomic orbitals of Ca contributed the most to HOMO, with lower and very close contributions of the atomic orbitals of O, Si and Cu, and no contribution of the atomic orbitals of P. The atomic orbitals contribution in LUMO showed the same behaviour. In Fig. 6d, the PDOS plot of BG FeO showed that the atomic orbitals of O and Si had close contribution to HOMO, with higher contribution by 3d and 4 s atomic orbitals of Fe and the highest contribution was by the 4 s atomic orbital of Ca.
In the PDOS plot of BG ZnO demonstrated Fig. 6e, the narrowest ΔE is clearly visualized, reflecting the highest reactivity of this structure compared with the other structures. The highest contribution to HOMO is offered by the atomic orbitals of Ca followed by closer contributions of those of Zn, O and Si, with no contribution from the atomic orbitals of P. The highest contribution for LUMO was from the atomic orbitals of Ca, followed by very close contributions from the atomic orbitals of Zn and Si, followed by O and finally P. The final PDOS plot presented in Fig. 6f for BG GeO indicated that the highest contribution for HOMO is given by the atomic orbitals of Ca, followed by very close contributions of the atomic orbitals of Ge, O and Si, and no contribution from P, and LUMO had the highest contribution from Ca, followed by Ge, then Si, O and the lowest contribution was from the atomic orbitals of P. Assessment of antibacterial activity. BG has been strongly advocated as a potential replacement for the graft materials currently in use 44 . According to reports, borate-based biomaterials were used at the infection site because of their antibacterial properties 45 .

Experimental FTIR band assignments.
Antibacterial activity was found in all of the tested composites as shown in Fig. 8 and Table 3. In the case of S. aureus shown in Fig. 8a, it was found that coating the glass composite particles with ZnO nanoparticles increased the antibacterial activity, being the maximum among the used MO by creating an inhibition zone of 50 mm. Coating with FeO, on the other hand, showed the minium activity, producing a 30 mm inhibition zone, which also reflects lower antibacterial activity towards S. aureus than BG control which created a 40 mm inhibition zone. Therefore, comparing the antibacterial activity of MO-coated BG based on the diameter of the inhibition zones shown in Table 3 confirmed that BG.ZnO demonstrated the highest activity, followed by BG control and BG.CuO (40 mm each), BG.GeO. (32 mm), and BG.FeO (30 mm).
The inhibition zones displayed in Fig. 8b also confirmed antibacterial activity against P. aeruginosa, where BG control and BG.ZnzO showed the highest antibacterial activity with inhibition zones of 35 and 30 mm, respectively. Furthermore, BG.CuO had higher antibacterial activity than BG.FeO, and BG.GeO which showed the lowest antibacterial activity.
In the case of A. hydrophila, all BG composition again showed antibacterial activity as shown in Fig. 8c. Similarly, BG control and BG.ZnO demonstrated the highest antibacterial activity based on their inhibition zones listed in Table 3. BG.FeO presented lower antibacterial activity followed by BG.GeO, then BG.CuO which present the lowest antibacterial activity against A. hydrophila. Composites inhibited the bacterial strains tested. This again could be due to the effect produced by the presence of boron and silicon ions present in the base composition of BG, as well as the additional effect posed by the MOs. The inhibition zones in the three bacterial strains S. aureus, P. aeruginosa, and A. hydrophila created by the different BG composite glass are demonstrated in Fig. 9  www.nature.com/scientificreports/ Theoretical infrared spectra, electronic properties and molecular electrostatic potential maps (MESP) were studied using DFT molecular modeling calculations at B3LYP/6-31 g(d) level. Theoretical infrared spectral were in perfect agreement with experimental data, reflecting high accuracy of computations. The reactivity of the different glass compositions was evaluated in terms of the electronic properties represented by total dipole moment (TDM), HOMO/LUMO band gap energy (ΔE) and MESP. All BG.MO structures demonstrated significant decrease in their TDM values compared to BG control, with ΔE also showing decrease in its value for all BG.MO composites. Within the four BG.MO composites, BG.ZnO is the most reactive structure owing to its highest TDM and lowest ΔE. MESP confirmed that oxygen atoms in the P 4 O 10 unit has higher electron density than phosphorus atoms. Oxygen atoms continued to show higher electronegativity with the presence of SiO 2 and CaO. Doping with CuO, FeO, ZnO and GeO resulted in significant change in the electron density distribution and electronegativity on the molecule's surface, introducing sites ready for nucleophilic attack and others ready for electrophilic attack.
The antibacterial activity of the BG control and the four BG.MO composites was assessed against S. aureus, P. aeruginosa and A. hydrophila pathogenic bacterial strains. All glass compositions showed antibacterial activity against the three pathogens, with BG.ZnO showing the highest antibacterial activity among the four BG.MO composites. This result is well correlated with molecular modeling results of reactivity and MESP owing to the highest reactivity of BG.ZnO, and the positive charge of ZnO nanoparticles in water suspensions, thus having